Benthic Heterotrophic Protist Communities of the Southern Baltic Analyzed with the Help of Curated Metabarcoding Studies

Abstract

Heterotrophic protists are key components of marine ecosystems. They act as controllers of bacterial and microphytobenthos production and contribute significantly to the carbon flux to higher trophic levels. Still, metabarcoding studies on benthic protist communities are much less frequent than for planktonic organisms. Especially in the Baltic Sea, representing the largest brackish water environment on earth, so far, no extensive metabarcoding studies have been conducted to assess the diversity of benthic protists in this unique and diverse habitat. This study aims to give first insights into the diversity of benthic protist communities in two different regions of the Baltic Sea, Fehmarnbelt, and Oderbank. Using amplicon sequencing of the 18S rDNA V9 region of over 100 individual sediment samples, we were able to show significant differences in the community composition between the two regions and to give insights into the vertical distribution of protists within the sediment (0-20 cm). The results indicate that the differences in community composition in the different regions might be explained by several abiotic factors such as salinity and water depth, but are also influenced by methodological aspects such as differences between DNA and RNA results.

Keywords: Baltic Sea; amplicon sequencing; brackish; diversity; sediment; unicellular eukaryotes.

Figure 1

(A) Sampling regions of the two cruises in the western Baltic Sea, (B) MUC core taken from sediments of the Fehmarnbelt region, (C) close up of the sampling stations in the Fehmarnbelt region during cruise EMB238, (D) close up of the sampling stations in the Oderbank region during cruise EMB267, (E) MUC core taken from sediments of the Oderbank region. Maps were created using Ocean Data View [26].

Figure 2

Rarefaction curves of samples from Fehmarnbelt and Oderbank region summed for sediment depth.

Figure 3

Results of metabarcoding studies of two regions in the western Baltic, Fehmarnbelt, and Oderbank. (A) Number of ASVs per station for Fehmarnbelt; (B) number of ASVs per sediment depth at Fehmarnbelt; (C) number of ASVs per station for Oderbank; (D) number of ASVs per sediment depth at Oderbank; (E) number of reads per station for Fehmarnbelt and (G) for Oderbank; (F) number of reads per sediment depth at Fehmarnbelt and (H) at Oderbank; (I) Shannon index per station at Fehmarnbelt and (K) at Oderbank; and (J) Shannon index per sediment depth at Fehmarnbelt and (L) at Oderbank.

Figure 4

Community composition of benthic protists at Fehmarnbelt and Oderbank showing the relative proportion of ASVs assigned to taxonomic groups. (A) Comparison of all samples from 0–2 cmbsf at Fehmarnbelt; (B) protist community structure obtained from depth profiles of cores (7 different depths, each with 3 replicates) at stations 17-6 and 18-6 at Fehmarnbelt; (C) vertical distribution of the protist community structure at Oderbank (summed for all stations from 5 different depth layers); (D) vertical changes in community structure for the different stations at Oderbank; and (E) direct comparison of the protist community structure of two stations from both regions, Fehmarnbelt and Oderbank, regarding different sediment layers based on RNA ASVs.

Figure 5

NMDS plot based on the Jaccard Index comparing benthic protist communities of the different stations in the western Baltic. (A) NMDS plot comparing all stations at Fehmarnbelt for the surface sediment layer 0–2 cmbsf. (B) Upset plot showing the number of shared ASVs between the different Fehmarnbelt stations (top bar chart) or unique to one station, as well as the overall number of ASVs (horizontal bars). Connected dots below the bar chart mean ASVs are shared between two or more stations. (C) Tree map showing the relative proportion of shared and unique ASVs per taxa group at Fehmarnbelt stations for the 0-2 cmbsf sediment layer. (D–F) Comparison of the vertical distribution of ASVs of the two Fehmarnbelt stations 17-6 and 18-6 in a similar manner as for (A–C). (G–I) Comparison of the community structure regarding all different sediment layers from stations at Oderbank analyzed in a similar manner as for (A–C).

Figure 6

NMDS plot based on the Jaccard distance comparing protist communities in different sediment depth layers of two sampling stations in the Oderbank region and two in the Fehmarnbelt region (A). (B) Upset plot showing the number of shared or unique ASVs for the different sampling depths for both regions (bar chart at the top). Connected dots below each bar show shared ASVs between different depths and stations. Horizontal bars indicate the total number of ASVs for the two stations in each region. (C) Tree map showing relative proportions of ASVs for taxa groups unique to one depth and region.

Figure 7

CCA analysis including a comparison of the benthic protist community composition based on ASVs including all stations of Fehmarnbelt and Oderbank regions analyzing the influence of water depth, sediment depth, and salinity.

Figure 8

List of genotypes obtained from species that were cultivated from samples collected from the two investigated regions. HFCC stands for the number of the Heterotrophic Flagellate Culture Collection Cologne. The red labeled data indicate that the genotype could not be recovered in metabarcoding studies of the respective region. The number shows the percentage of samples in which they were detected in a regional dataset.